Figure 4. Dynamic reorganization of the ERM network is important for microcluster formation. (A) DT40 B cells expressing Ezrin-WT–GFP were settled onto planar lipid bilayers containing antigen (Ag) and visualized by TIRFM. GFP and antigen images are pseudocolored according to the lookup tables on the left. Images in the right panels are magnified time sequence images of the left panel (boxed areas) highlighting regions of ezrin (dotted red lines) and antigen (dotted yellow lines). White ovals indicate antigen microcluster and corresponding location with respect to ezrin (black ovals). (B) Representative images of antigen microclusters in DT40 B cells expressing Ezrin-WT, Ezrin-CA, or Ezrin-DN on planar lipid bilayers. (C) Quantification of the mean intensity of antigen microclusters in cells expressing Ezrin-WT (625.4 ± 66.2 arbitrary units [AU]), Ezrin-CA (769.1 ± 37.9 AU), and Ezrin-DN (267.2 ± 95.9 AU). ***, P < 0.001. (D) Quantification of the area of antigen microclusters in cells expressing Ezrin-WT (0.204 ± 0.006 µm), Ezrin-CA (0.265 ± 0.013 µm), and Ezrin-DN (0.017 ± 0.006 µm). ***, P = 0.0004. (E) Quantification of the number of antigen microclusters in cells expressing Ezrin-WT (58 ± 3), Ezrin-CA (37 ± 3), and Ezrin-DN (34 ± 5). **, P = 0.002; ***, P = 0.0003. (F) Quantification of the total antigen intensity at the contact in cells expressing Ezrin-WT (5.4 × 106 ± 6.1 × 105 AU), Ezrin-CA (2.8 × 106 ± 2.7 × 105 AU), and Ezrin-DN (1.2 × 106 ± 1.5 × 105 AU). ***, P < 0.0001. Data are representative of at least two experiments with the mean ± SEM indicated in red. Between 16 and 28 cells were quantified in each cell type, representing a total of 2,298 microclusters. (G and H) Cells expressing WT and mutated ezrin were settled onto anti-IgM–coated plates for the indicated time. Cells were lysed and analyzed by SDS-PAGE followed by immunoblotting with anti–phospho-p44 and -p42 MAPK (Erk1 and Erk2) or antitubulin. (I and J) Comparison of the normalized intensity of pERK upon stimulation in cells expressing Ezrin-CA–GFP (I) or Ezrin-DN–GFP (J) with WT Ezrin-GFP. Data are representative of at least two independent experiments. Bars, 2 µm.
Figure 4.

Dynamic reorganization of the ERM network is important for microcluster formation. (A) DT40 B cells expressing Ezrin-WT–GFP were settled onto planar lipid bilayers containing antigen (Ag) and visualized by TIRFM. GFP and antigen images are pseudocolored according to the lookup tables on the left. Images in the right panels are magnified time sequence images of the left panel (boxed areas) highlighting regions of ezrin (dotted red lines) and antigen (dotted yellow lines). White ovals indicate antigen microcluster and corresponding location with respect to ezrin (black ovals). (B) Representative images of antigen microclusters in DT40 B cells expressing Ezrin-WT, Ezrin-CA, or Ezrin-DN on planar lipid bilayers. (C) Quantification of the mean intensity of antigen microclusters in cells expressing Ezrin-WT (625.4 ± 66.2 arbitrary units [AU]), Ezrin-CA (769.1 ± 37.9 AU), and Ezrin-DN (267.2 ± 95.9 AU). ***, P < 0.001. (D) Quantification of the area of antigen microclusters in cells expressing Ezrin-WT (0.204 ± 0.006 µm), Ezrin-CA (0.265 ± 0.013 µm), and Ezrin-DN (0.017 ± 0.006 µm). ***, P = 0.0004. (E) Quantification of the number of antigen microclusters in cells expressing Ezrin-WT (58 ± 3), Ezrin-CA (37 ± 3), and Ezrin-DN (34 ± 5). **, P = 0.002; ***, P = 0.0003. (F) Quantification of the total antigen intensity at the contact in cells expressing Ezrin-WT (5.4 × 106 ± 6.1 × 105 AU), Ezrin-CA (2.8 × 106 ± 2.7 × 105 AU), and Ezrin-DN (1.2 × 106 ± 1.5 × 105 AU). ***, P < 0.0001. Data are representative of at least two experiments with the mean ± SEM indicated in red. Between 16 and 28 cells were quantified in each cell type, representing a total of 2,298 microclusters. (G and H) Cells expressing WT and mutated ezrin were settled onto anti-IgM–coated plates for the indicated time. Cells were lysed and analyzed by SDS-PAGE followed by immunoblotting with anti–phospho-p44 and -p42 MAPK (Erk1 and Erk2) or antitubulin. (I and J) Comparison of the normalized intensity of pERK upon stimulation in cells expressing Ezrin-CA–GFP (I) or Ezrin-DN–GFP (J) with WT Ezrin-GFP. Data are representative of at least two independent experiments. Bars, 2 µm.

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